Pbmc megakaryocte cell pulldown
Kent Riemondy RBI
R Sys.Date()
Examine Lna pulldown data
Organize single cell libraries
First designate the libraries and the cells that were subsampled.
cells <- list(
mkcell_pulldown = c("TGCGCAGCAGGTCGTC",
"ACTTGTTAGGACCACA",
"CCATTCGTCCCTGACT",
"TGTCCCAGTAAACACA"))
libs <- c(
"kirkpatrick",
"mkcell_pulldown")
bc_metadat <- read_tsv(file.path(data_dir,
"kirkpatrick",
"fastq",
"original",
"barcodes_from_10x_run.txt"),
col_names = c("cell_id", "barcode_10x"))
## original library to compare against
reflib <- "kirkpatrick"
## all subsampled libs to plot
subsampled_libs <- "mkcell_pulldown"
## reference subsampled lib for subsampled vs control plots
subsampled_lib <- "mkcell_pulldown"Load and organize a table for each library of read counts per cell per gene, and a table of umi counts per cell per gene.
umis_to_genes <- function(umipath, cells_to_exclude = c("Cell_unmatched")){
umis <- read_tsv(umipath,
col_names = c("barcode_10x",
"umi_molecule",
"count")) %>%
filter(barcode_10x != cells_to_exclude)
mol_fields <- str_count(umis$umi_molecule[1], "::")
if(mol_fields == 2 ){
umis <- separate(umis, umi_molecule,
into = c("seq", "genome", "gene"),
sep = "::") %>%
mutate(gene = str_c(genome, "::", gene))
} else if (mol_fields == 1){
umis <- separate(umis, umi_molecule,
into = c("seq", "gene"),
sep = "::")
} else {
stop("separator :: missing from umi_molecule field")
}
reads <- select(umis,
barcode_10x,
gene,
count)
reads <- group_by(reads,
barcode_10x, gene) %>%
summarize(counts = sum(count))
reads <- spread(reads, barcode_10x, counts,
fill = 0L)
reads
}
## read in umigroups flat file with read counts per umi per gene per cell
## expand out to a read count matrix
umipaths <- file.path(data_dir,
libs,
"umis",
"umigroups.txt.gz")
read_dat <- map(umipaths,
~umis_to_genes(.))
names(read_dat) <- libs
## read in umi_tools count table with umi counts per gene per cell
umi_dat <- map(libs,
~read_tsv(file.path(data_dir,
.x,
"dgematrix",
"dge_matrix.txt")))
names(umi_dat) <- libs
cell_obj_mdata <- map(cells,
~mutate(bc_metadat,
subsampled = ifelse(barcode_10x %in% .x,
TRUE,
FALSE)))Next organize these tables into simple classes called subsampled-sets to keep track of each experiment’s relavant raw, processed, and meta data.
#' simple class to hold info for each experiment
create_sc_obj <- function(umi_df,
read_df,
cell_mdata_df){
x <- list()
class(x) <- "subsampled-set"
x$umis <- umi_df
x$reads <- read_df
x$meta_dat <- cell_mdata_df
return(x)
}
sc_objs <- list(umi_dat, read_dat, cell_obj_mdata)
sc_objs <- pmap(sc_objs, create_sc_obj)
rm(umi_dat)
rm(read_dat)Next perform basic processing. 1) generate separate objects to store sparse matrices of umi and read counts. 2) normalize read and umi count data by total library size (sum of all read or umi counts for all cells in the experiment) and report as Reads per million or UMIs per million. 3) Compute per cell metrics (read and umi counts, sequencing saturation)
tidy_to_matrix <- function(df){
df <- as.data.frame(df)
rownames(df) <- df[, 1]
df[, 1] <- NULL
mat <- as.matrix(df)
mat <- as(mat, "sparseMatrix")
return(mat)
}
#' keep both tidy and matrix objs
generate_matrices <- function(sc_obj){
sc_obj$umi_matrix <- tidy_to_matrix(sc_obj$umis)
sc_obj$read_matrix <- tidy_to_matrix(sc_obj$reads)
sc_obj
}
#' normalize by library size (Reads per Million)
norm_libsize <- function(sc_obj){
sc_obj$norm_umi <- 1e6 * sweep(sc_obj$umi_matrix, 2,
sum(as.vector(sc_obj$umi_matrix)), "/")
sc_obj$norm_reads <- 1e6 * sweep(sc_obj$read_matrix, 2,
sum(as.vector(sc_obj$read_matrix)), "/")
sc_obj
}
add_metadata <- function(sc_obj, dat){
if (is.vector(dat)){
new_colname <- deparse(substitute(dat))
df <- data_frame(!!new_colname := dat)
df[[new_colname]] <- dat
df[["cell_id"]] <- names(dat)
sc_obj$meta_dat <- left_join(sc_obj$meta_dat,
df,
by = "cell_id")
} else if (is.data.frame(dat)) {
sc_obj$meta_dat <- left_join(sc_obj$meta_dat,
dat,
by = "cell_id")
}
sc_obj
}
compute_summaries <- function(sc_obj){
## raw counts
total_umis <- colSums(sc_obj$umi_matrix)
names(total_umis) <- colnames(sc_obj$umi_matrix)
total_reads <- colSums(sc_obj$read_matrix)
names(total_reads) <- colnames(sc_obj$read_matrix)
## norm counts
norm_total_umis <- colSums(sc_obj$norm_umi)
names(norm_total_umis) <- colnames(sc_obj$norm_umi)
norm_total_reads <- colSums(sc_obj$norm_reads)
names(norm_total_reads) <- colnames(sc_obj$norm_reads)
sc_obj <- add_metadata(sc_obj, total_umis)
sc_obj <- add_metadata(sc_obj, total_reads)
sc_obj <- add_metadata(sc_obj, norm_total_umis)
sc_obj <- add_metadata(sc_obj, norm_total_reads)
## compute cDNA duplication rate
sc_obj$meta_dat$cDNA_duplication <- 1 - (sc_obj$meta_dat$total_umis /
sc_obj$meta_dat$total_reads)
sc_obj
}
sc_objs <- map(sc_objs, generate_matrices)
sc_objs <- map(sc_objs, norm_libsize)
sc_objs <- map(sc_objs, compute_summaries)Compute enrichment of reads/umis over the original library.
sc_objs <- map(sc_objs,
function(sub_dat){
og_counts <- select(sc_objs[[reflib]]$meta_dat,
og_total_reads = total_reads,
og_total_umis = total_umis,
og_norm_total_umis = norm_total_umis,
og_norm_total_reads = norm_total_reads,
og_cDNA_duplication = cDNA_duplication,
cell_id)
sub_dat$meta_dat <- left_join(sub_dat$meta_dat,
og_counts,
by = "cell_id")
sub_dat$meta_dat <- mutate(sub_dat$meta_dat,
read_proportion = log2( total_reads / og_total_reads),
umi_proportion = log2( total_umis / og_total_umis),
norm_read_proportion = log2( norm_total_reads /
og_norm_total_reads),
norm_umi_proportion = log2( norm_total_umis /
og_norm_total_umis))
sub_dat
})plot cDNA duplication rate
sc_metadat <- map(sc_objs, ~.x$meta_dat) %>%
bind_rows(.id = "library")
ggplot(sc_metadat, aes(total_reads, cDNA_duplication)) +
geom_point(aes(color = subsampled)) +
labs(x = expression(paste("Read count (log"[10],")")),
y = "Sequencing saturation") +
scale_x_log10() +
scale_color_manual(values = color_palette) +
facet_wrap(~library)
Note the high level of sequencing saturation (0 = no-duplication, 1 = all duplicates) in the original library. Also note that the libraries tend to have higher saturatioin rates, after subsampling.
plot read and umi counts per library
global_plot_theme <- theme(
legend.position = "top",
legend.text = element_text(size = 10),
strip.text = element_text(size = 8))
subsampled_metadat <- sc_metadat[sc_metadat$library != reflib, ] %>%
mutate(library = factor(library,
levels = c(subsampled_libs)))
unnorm_plt <- ggplot(subsampled_metadat,
aes(og_total_reads / 3, total_reads / 3, colour = subsampled)) +
geom_point(size = 0.5) +
geom_abline(slope = 1) +
facet_wrap(~library, nrow = 1) +
# coord_fixed() +
xlab("original library\nreads count (Thousands)") +
ylab("subsampled library\nreads count (Thousands)") +
# ggtitle("Raw reads associated with each cell barcode") +
scale_colour_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 10, line_size = .5) +
theme(aspect.ratio = 1) +
global_plot_theme
norm_plt <- ggplot(subsampled_metadat, aes(og_norm_total_reads / 1e3,
norm_total_reads / 1e3,
colour = subsampled)) +
geom_point(size = 0.5) +
geom_abline(slope = 1) +
facet_wrap(~library, nrow = 1) +
xlab("original library\nRPM (Thousands)") +
ylab("subsampled library\nRPM (Thousands)") +
scale_colour_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 10, line_size = 0.5) +
theme(aspect.ratio = 1) +
global_plot_theme
unnorm_umi_plt <- ggplot(subsampled_metadat,
aes(og_total_umis / 1e3, total_umis / 1e3, colour = subsampled)) +
geom_point(size = 0.5) +
geom_abline(slope = 1) +
# coord_fixed() +
facet_wrap(~library, nrow = 1) +
xlab("original library\nUMI count (Thousands)") +
ylab("subsampled library\nUMI count (Thousands)") +
scale_colour_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 10, line_size = .5) +
theme(aspect.ratio = 1) +
global_plot_theme
norm_umi_plt <- ggplot(subsampled_metadat,
aes(og_norm_total_umis / 1e3, norm_total_umis / 1e3, colour = subsampled)) +
geom_point(size = 0.5) +
geom_abline(slope = 1) +
facet_wrap(~library, nrow = 1) +
xlab("original library\nUMI normalized RPM (Thousands)") +
ylab("subsampled library\nUMIs per Million (Thousands)") +
scale_colour_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 10, line_size = 0.5) +
theme(aspect.ratio = 1) +
global_plot_theme
plt <- plot_grid(unnorm_plt, norm_plt, unnorm_umi_plt, norm_umi_plt,
labels = "AUTO",
align = 'hv')
plt
save_plot("reads_per_barcode_scatterplots.pdf", plt, nrow = 2, ncol = 2, base_width = 8 )Plot enrichment of reads/umis
read <- ggplot(subsampled_metadat,
aes(cell_id, norm_read_proportion, colour = subsampled)) +
geom_point() +
labs(x = "Cell",
y = expression(paste( " Log"[2], " normalized reads ", frac("subsampled", "original")))) +
scale_colour_manual(name = "subsampled:", values = color_palette) +
facet_wrap(~library, nrow = 1) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12))
umi <- ggplot(subsampled_metadat,
aes(cell_id, norm_umi_proportion, colour = subsampled)) +
geom_point() +
labs(x = "Cell",
y = expression(paste( "Log"[2], " normalized UMIs ", frac("subsampled", "original")))) +
scale_colour_manual(name = "subsampled:", values = color_palette) +
facet_wrap(~library, nrow = 1) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12))
plt <- plot_grid(read, umi,
labels = "AUTO",
align = 'hv')
plt
ggsave("reads_umi_ratio_per_barcode_normalized.pdf", width = 8, height = 5)
umi <- ggplot(filter(subsampled_metadat, library %in% subsampled_libs),
aes(cell_id, norm_umi_proportion, colour = subsampled)) +
geom_point() +
labs(x = "Cell",
y = expression(paste( "Log"[2], " normalized UMIs ", frac("subsampled", "original")))) +
scale_colour_manual(name = "subsampled:", values = color_palette) +
facet_wrap(~library, nrow = 1) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12))
ggsave("umi_ratio_MA_normalized.pdf", umi, width =5, height = 5)
umi <- ggplot(filter(subsampled_metadat, library %in% subsampled_libs),
aes(og_total_umis, umi_proportion, colour = subsampled)) +
geom_point() +
labs(x = "Total UMIs per barcode",
y = expression(paste( "Log"[2], " UMIs ", frac("subsampled", "original")))) +
scale_colour_manual(name = "subsampled:", values = color_palette) +
facet_wrap(~library, nrow = 1) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(legend.position = "top",
legend.text = element_text(size = 12))
ggsave("umi_ratio_MA.pdf", umi, width =5, height = 5)ggplot(subsampled_metadat, aes(subsampled,
read_proportion, fill = subsampled)) +
geom_boxplot() +
facet_wrap(~library, nrow = 1) +
xlab("Selected for Reamplification") +
ylab(expression(paste( "Log"[2], " Reads ", frac("subsampled", "original")))) +
scale_fill_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 16, line_size = 0.5) +
theme(
axis.title.x = element_blank(),
axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12)
)
ggsave("reads_ratio_per_barcode_boxplot.pdf", width = 6, height = 5)
ggplot(subsampled_metadat, aes(subsampled,
umi_proportion, fill = subsampled)) +
geom_boxplot() +
facet_wrap(~library, nrow = 1) +
xlab("Selected for Reamplification") +
ylab(expression(paste( "Log"[2], " UMIs ", frac("subsampled", "original")))) +
scale_fill_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 16, line_size = 0.5) +
theme(
axis.title.x = element_blank(),
axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12)
)
ggsave("umi_ratio_per_barcode_boxplot.pdf", width = 6, height = 5)
ggplot(subsampled_metadat, aes(subsampled,
norm_read_proportion, fill = subsampled)) +
geom_boxplot() +
facet_wrap(~library, nrow = 1) +
xlab("Selected for Reamplification") +
ylab(expression(paste( "Log"[2], " normalized Reads ", frac("subsampled", "original")))) +
scale_fill_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 16, line_size = 0.5) +
theme(
axis.title.x = element_blank(),
axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12)
)
ggsave("norm_reads_ratio_per_barcode_boxplot.pdf", width = 6, height = 5)
ggplot(subsampled_metadat, aes(subsampled,
norm_umi_proportion, fill = subsampled)) +
geom_boxplot() +
facet_wrap(~library, nrow = 1) +
xlab("Selected for Reamplification") +
ylab(expression(paste( "Log"[2], " normalized UMIs ", frac("subsampled", "original")))) +
scale_fill_manual(name = "subsampled:",
values = color_palette) +
theme_cowplot(font_size = 16, line_size = 0.5) +
theme(
axis.title.x = element_blank(),
axis.text.x = element_blank(),
legend.position = "top",
legend.text = element_text(size = 12)
)
ggsave("norm_umi_ratio_per_barcode_boxplot.pdf", width = 6, height = 5)dat <- group_by(subsampled_metadat, library) %>%
filter(library != reflib) %>%
mutate(total_new = sum(total_reads, na.rm = T),
total_old = sum(og_total_reads, na.rm = T))
dat_group <- group_by(dat, library, subsampled) %>%
summarize(total_new = sum(total_reads,
na.rm = T) / unique(total_new),
total_old = sum(og_total_reads,
na.rm = T) / unique(total_old)) %>%
gather(lib, percent_lib, -library, -subsampled ) %>%
mutate(lib = factor(lib, levels = c("total_old", "total_new"),
labels = c("original\nlibrary", "subsampled\nlibrary")))
ggplot(dat_group, aes(lib, percent_lib, fill = subsampled)) +
geom_bar(stat = "identity") +
ylab("Fraction of\n Reads Assigned") +
scale_fill_manual(name = "subsampled:",
values = color_palette) +
facet_wrap(~library) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(
axis.title.x = element_blank(),
axis.text.x = element_text(angle = 90, hjust = 0.5, vjust = 0.5),
legend.position = "top",
legend.text = element_text(size = 16)
)
ggsave("proportion_reads_all_barcode_barplot.pdf", width = 7, height = 7)
dat_group %>%
rename(Method = library) %>%
spread(lib, percent_lib) %>%
mutate(`Targeted Library Read Fold-Enrichment` = `subsampled\nlibrary` / `original\nlibrary`) %>%
filter(subsampled == T) %>%
select(Method, `Targeted Library Read Fold-Enrichment`)Genes detected
## compute per gene or per gene/umi combo enrichment
detected_molecules <- function(sc_obj, molecule = "gene"){
umis <- sc_obj$umi_matrix
if (molecule == "gene"){
n_genes <- colSums(umis > 0)
out_mdat <- data_frame(cell_id = colnames(umis),
n_genes = n_genes)
sc_obj <- add_metadata(sc_obj, out_mdat)
}
}
sc_objs <- map(sc_objs, ~detected_molecules(.x))subsampled_metadat <- map(sc_objs, ~.x$meta_dat) %>%
bind_rows(.id = "library") %>%
mutate(library = factor(library,
levels = libs))
og_genes <- filter(subsampled_metadat,
library == reflib) %>%
dplyr::select(cell_id,
og_genes = n_genes)
subsampled_metadat <- left_join(subsampled_metadat,
og_genes,
by = "cell_id") %>%
filter(library != reflib)
ggplot(subsampled_metadat, aes(og_genes,
n_genes, colour = subsampled)) +
geom_point() +
ylab("subsampled_genes") +
xlab("original_genes") +
scale_colour_manual(name = "subsampled:", values = color_palette) +
facet_wrap(~library, nrow = 1) +
theme_cowplot(font_size = 16, line_size = 1) +
theme(
legend.position = "top",
legend.text = element_text(size = 16)
)
Parse out new versus previously identified genes
calc_gene_sensitivity <- function(sc_obj,
type = "umi"){
if (type == "umi"){
count_matrix <- sc_obj$umi_matrix
} else {
count_matrix <- sc_obj$read_matrix
}
# generate list named with barcode of each detected gene and
# respective read/umi count
genes_detected <- apply(count_matrix, 2, function(x) x[x > 0])
sc_obj$genes_detected <- genes_detected
sc_obj
}
sc_objs <- map(sc_objs, calc_gene_sensitivity)sc_objs <- map(sc_objs,
function(x){
og_genes <- sc_objs[[reflib]]$genes_detected
sub_genes <- x$genes_detected
# subset list of cell barcodes to the same as the og experiment
# and also reorders the barcodes to match
sub_genes <- sub_genes[names(og_genes)]
if(length(sub_genes) != length(og_genes)){
stop("barcode lengths not the same")
}
shared_genes <- map2(sub_genes,
og_genes,
~intersect(names(.x),
names(.y)))
new_genes <- map2(sub_genes,
og_genes,
~setdiff(names(.x),
names(.y)))
not_recovered_genes <- map2(og_genes,
sub_genes,
~setdiff(names(.x),
names(.y)))
x$shared_genes <- shared_genes
x$new_genes <- new_genes
x$not_recovered_genes <- not_recovered_genes
return(x)
})
## add gene recovery info to meta data table
sc_objs <- map(sc_objs,
function(x){
shared_genes <- map2_dfr(x$shared_genes,
names(x$shared_genes),
function(x, y){
data_frame(cell_id = y,
shared_genes = length(x))
})
not_recovered_genes <- map2_dfr(x$not_recovered_genes,
names(x$not_recovered_genes),
function(x, y){
data_frame(cell_id = y,
not_recovered_genes = length(x))
})
new_genes <- map2_dfr(x$new_genes,
names(x$new_genes),
function(x, y){
data_frame(cell_id = y,
new_genes = length(x))
})
gene_mdata <- left_join(shared_genes,
not_recovered_genes,
by = "cell_id") %>%
left_join(., new_genes, by = "cell_id")
x <- add_metadata(x, gene_mdata)
x
})
subsampled_metadat <- map(sc_objs, ~.x$meta_dat) %>%
bind_rows(.id = "library") %>%
mutate(library = factor(library,
levels = libs))genes_recovered <- subsampled_metadat %>%
dplyr::filter(library != reflib) %>%
dplyr::select(cell_id,
library,
subsampled,
shared_genes,
not_recovered_genes,
new_genes)
genes_recovered <- gather(genes_recovered,
key = type, value = count,
-cell_id, -subsampled, -library)
genes_recovered <- mutate(genes_recovered,
type = str_replace_all(type, "_", "\n"))
plt <- ggplot(genes_recovered,
aes(cell_id, count)) +
geom_point(aes(color = subsampled),
size = 0.6,
alpha = 0.75) +
facet_grid(type ~ library) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
strip.text.y = element_text(size = 12,
margin = margin(0,0.2,0,0.2, "cm"))) +
scale_color_manual(values = color_palette)
plt 
save_plot("new_genes_detected.pdf", plt, base_width = 8, base_height = 8)
targeted <- genes_recovered %>%
dplyr::filter(library %in% subsampled_libs,
subsampled)
targetedplt_dat <- genes_recovered %>%
dplyr::filter(!library %in% subsampled_libs,
subsampled) %>%
group_by(type) %>%
summarize(count = mean(count)) %>%
mutate(cell_id = "Not Targeted Barcodes") %>%
bind_rows(targeted, .) %>%
filter(type == "new\ngenes")
plt <- ggplot(plt_dat,
aes(cell_id, count)) +
geom_bar(aes(fill = cell_id),
stat = "identity") +
labs(y = "Newly detected genes") +
theme(axis.title.x = element_blank(),
axis.text.x = element_text(angle = 45,
hjust = 1),
legend.position = "none") +
scale_fill_brewer(palette = "Set1")
plt
save_plot("new_genes_barplot.pdf", plt,
base_width = 3.6, base_height = 5)Plot expression per cell
MA plots
calc_ma <- function(xmat, ymat, cell = "GCAGTTAAGTGTCCAT"){
x_rn <- rownames(xmat)
y_rn <- rownames(ymat)
xmat <- log2(xmat + 1)
ymat <- log2(ymat + 1)
rownames(xmat) <- x_rn
rownames(ymat) <- y_rn
m <- rowMeans(log2(((2^ymat + 2^xmat) / 2) + 1))
a <- xmat[, cell] - ymat[, cell]
data_frame(gene = names(a),
mean_expression_log2 = m,
log2_diff = a)
}
genes_to_plot <- rownames(sc_objs$mkcell_pulldown$umi_matrix)
cols <- colnames(sc_objs[[subsampled_lib]]$norm_umi)
cell_ids <- str_c(cells[[subsampled_lib]], "-1")
## append genes to reference library if necessary
ref_mat <- standardize_rows(sc_objs[[subsampled_lib]]$umi_matrix[, cols],
sc_objs[[reflib]]$umi_matrix[, cols])
ma_dat <- map(cell_ids,
~calc_ma(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot, cols],
ref_mat[genes_to_plot, cols],
cell = .x))
names(ma_dat) <- cell_ids
ma_dat <- bind_rows(ma_dat, .id = "cell")
plot_ma <- function(df){
n_up <- filter(df, log2_diff > 0) %>%
group_by(cell) %>%
summarize(n = n(), n = paste0("up = ", n))
n_down <- filter(df, log2_diff < 0) %>%
group_by(cell) %>%
summarize(n = n(), n = paste0("down = ", n))
if (nrow(n_down) == 0) {
n_down = data_frame(cell = df$cell %>% unique(),
n = "down = 0")
}
plt <- ggplot(df,
aes(mean_expression_log2,
log2_diff)) +
geom_hline(aes(yintercept = 0), linetype = "dashed", colour = "grey") +
geom_point(size = 0.25) +
geom_text(data = n_up, aes(x = max(ma_dat$mean_expression_log2) * 0.9,
y = max(ma_dat$log2_diff) * 1.2,
label = n)) +
geom_text(data = n_down, aes(x = max(ma_dat$mean_expression_log2) * 0.9,
y = min(ma_dat$log2_diff) * 1.2,
label = n)) +
facet_wrap(~cell) +
labs(x = expression(paste("Abundance (log"[2], ")")),
y = expression(paste(frac("Subsampled","Original"), " (log"[2], ")")))
plt
}
plt <- plot_ma(ma_dat)
save_plot("per_cell_MA_plot_all_genes.pdf", plt, base_height = 6)
## Shared genes
ma_dat <- map(cell_ids,
function(x){
genes_to_plot <- sc_objs[[subsampled_lib]]$shared_genes[[x]]
calc_ma(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot,
cols],
ref_mat[genes_to_plot, cols],
cell = x)
})
names(ma_dat) <- cell_ids
ma_dat <- bind_rows(ma_dat, .id = "cell")
plt <- plot_ma(ma_dat)
save_plot("per_cell_MA_plot_shared_genes.pdf", plt, base_height = 6)
## New genes
ma_dat <- map(cell_ids,
function(x){
genes_to_plot <- sc_objs[[subsampled_lib]]$new_genes[[x]]
calc_ma(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot,
cols],
ref_mat[genes_to_plot, cols],
cell = x)
})
names(ma_dat) <- cell_ids
ma_dat <- bind_rows(ma_dat, .id = "cell")
plt <- plot_ma(ma_dat)
save_plot("per_cell_MA_plot_new_genes.pdf", plt, base_height = 6)Histograms
get_expr <- function(xmat, ymat, cell = "GCAGTTAAGTGTCCAT"){
xmat <- log2(xmat[, cell] + 1)
ymat <- log2(ymat[, cell] + 1)
data_frame(subsampled = xmat,
original = ymat) %>%
gather(library,
Expression)
}
plot_histogram <- function(df){
ggplot(df,
aes_string("Expression")) +
geom_density(aes_string(fill = "library"),
alpha = 0.66) +
scale_fill_viridis_d(name = "") +
facet_wrap(~cell, nrow = 1) +
theme(legend.position = "top",
strip.text = element_text(size = 8))
}
expr_dat <- map(cell_ids,
function(x){
genes_to_plot <- names(sc_objs[[subsampled_lib]]$genes_detected[[x]])
get_expr(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot,
cols],
ref_mat[genes_to_plot, cols],
cell = x)
})
names(expr_dat) <- cell_ids
expr_dat <- bind_rows(expr_dat, .id = "cell")
expressed_in_subsampled_plt <- plot_histogram(expr_dat)
expr_dat <- map(cell_ids,
function(x){
genes_to_plot <- sc_objs[[subsampled_lib]]$shared_genes[[x]]
get_expr(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot,
cols],
ref_mat[genes_to_plot, cols],
cell = x)
})
names(expr_dat) <- cell_ids
expr_dat <- bind_rows(expr_dat, .id = "cell")
share_genes_plt <- plot_histogram(expr_dat)
expr_dat <- map(cell_ids,
function(x){
genes_to_plot <- sc_objs[[subsampled_lib]]$new_genes[[x]]
get_expr(sc_objs[[subsampled_lib]]$umi_matrix[genes_to_plot,
cols],
ref_mat[genes_to_plot, cols],
cell = x)
})
names(expr_dat) <- cell_ids
expr_dat <- bind_rows(expr_dat, .id = "cell")
new_gene_plt <- plot_histogram(expr_dat)
plts <- list(
expressed_in_subsampled_plt,
share_genes_plt,
new_gene_plt
)
plt <- plot_grid(plotlist = plts, ncol = 1)
plt
save_plot("expression_histograms.pdf", plt,
ncol = 1, nrow = 3,
base_height = 4,
base_aspect_ratio = 2)UMIs detected
Parse out new verses old UMIs
compare_umis <- function(path_to_ctrl,
path_to_test,
return_summary = F,
cells_exclude = "Cell_unmatched"){
## umi seqs should be produced by ./get_molecule_info
ctrl_umi_seqs <- read_tsv(path_to_ctrl,
col_names = c("barcode_10x",
"umi_molecule",
"count")) %>%
filter(!barcode_10x %in% cells_exclude)
test_umi_seqs <- read_tsv(path_to_test,
col_names = c("barcode_10x",
"umi_molecule",
"count")) %>%
filter(!barcode_10x %in% cells_exclude)
umi_seqs <- full_join(ctrl_umi_seqs,
test_umi_seqs,
by = c("barcode_10x", "umi_molecule"))
if (return_summary) {
umi_seqs %>%
mutate(new_umi = ifelse(is.na(count.x) & !is.na(count.y),
1L,
0L),
not_detected_umi = ifelse(!is.na(count.x) & is.na(count.y),
1L,
0L),
shared_umi = ifelse(!is.na(count.x) & !is.na(count.y),
1L,
0L)) %>%
group_by(barcode_10x) %>%
summarize(new_umis = sum(new_umi),
not_detected_umis = sum(not_detected_umi),
shared_umis = sum(shared_umi))
} else {
umi_seqs
}
}
umi_files <- file.path(data_dir, libs, "umis", "umigroups.txt.gz")
umi_summaries <- map(umi_files[2],
~compare_umis(umi_files[1], .x, return_summary = T))
names(umi_summaries) <- umi_files[2] %>%
str_split(., "/") %>%
map_chr(~.x[7])
umi_summary <- bind_rows(umi_summaries, .id = "library")
umis_recovered <- umi_summary %>%
gather(class, count, -barcode_10x, -library)
## annotate with subsampled or not
cell_annot <- data_frame(barcode_10x = cells,
library = libs,
subsampled = T) %>%
unnest()
umis_recovered <- umis_recovered %>%
mutate(subsampled = ifelse(str_replace(barcode_10x, "-1", "") %in% unlist(cells),
T,
F)) %>%
arrange(subsampled)
plt <- ggplot(umis_recovered,
aes(barcode_10x, count)) +
geom_point(aes(colour = subsampled),
size = 0.6,
alpha = 0.75) +
facet_grid(library ~ class) +
theme(axis.text.x = element_blank(),
axis.title.x = element_blank(),
strip.text.y = element_text(size = 12,
margin = margin(0,0.2,0,0.2, "cm"))) +
scale_color_manual(values = color_palette)
plt 
umi_seqs <- map(umi_files[2],
~compare_umis(umi_files[1], .x, return_summary = F))
names(umi_seqs) <- umi_files[2] %>%
str_split(., "/") %>%
map_chr(~.x[7])
new_umis <- map(umi_seqs,
~filter(.x,
str_replace(barcode_10x, "-1", "") %in% unlist(cells),
!is.na(count.y),
is.na(count.x)) %>%
separate(umi_molecule, c("seq", "gene"), sep = "::") %>%
select(-starts_with("count")))
old_umis <- map(umi_seqs,
~filter(.x,
str_replace(barcode_10x, "-1", "") %in% unlist(cells),
!is.na(count.x),
!is.na(count.y)) %>%
separate(umi_molecule, c("seq", "gene"), sep = "::") %>%
select(-starts_with("count")))
umi_edit_dist <- map2(new_umis,
old_umis,
~left_join(.x, .y,
by = c("barcode_10x", "gene")) %>%
na.omit() %>%
mutate(ed = kentr::get_hamming_pairs(seq.x, seq.y)) %>%
group_by(barcode_10x, seq.y, gene) %>%
summarize(min_ed = min(ed)) %>%
ungroup())
umi_edit_dist <- bind_rows(umi_edit_dist,
.id = "library")
ggplot(umi_edit_dist, aes(barcode_10x,
min_ed)) +
geom_boxplot(coef = Inf) +
facet_wrap(~library, scales = "free_x") +
labs(y = "Minimum edit distance\noriginal vs. new UMIs") +
theme(axis.text.x = element_text(angle = 90,
hjust = 1,
vjust = 0.5),
axis.title.x = element_blank())
rm(umi_seqs)tSNE analysis
original library tSNE
library(Seurat)
mat <- sc_objs[[reflib]]$umi_matrix
sobj <- CreateSeuratObject(mat)
sobj <- NormalizeData(sobj)
sobj <- ScaleData(sobj, vars.to.regress = "nUMI")
sobj <- FindVariableGenes(sobj, do.plot = F)
sobj <- RunPCA(sobj, pc.genes = sobj@var.genes, pcs.compute = 20, do.print = F)
sobj <- RunTSNE(sobj, dims.use = 1:18, seed.use = 20180516)
sobj <- FindClusters(sobj, dims.use = 1:18, resolution = 0.6,
print.output = F,
random.seed = 20180516)plt <- TSNEPlot(sobj,
colors.use = brewer.pal(12, "Paired"),
do.label = T) +
labs(title = "PBMCs") +
theme(legend.position = "none")
plt
save_plot("original_mkcells_tsne.pdf", plt, base_height = 4.25, base_width = 4.25)original library tSNE supplemented with resampled barcodes
mat <- sc_objs[[reflib]]$umi_matrix
subsampled_ids <- sc_objs[[subsampled_libs]]$meta_dat %>%
filter(subsampled) %>%
pull(cell_id)
subsampled_mat <- sc_objs[[subsampled_libs]]$umi_matrix[, subsampled_ids]
colnames(subsampled_mat) <- str_c(colnames(subsampled_mat), "::", "subsampled")
mat <- as.data.frame(as.matrix(mat)) %>% rownames_to_column("gene")
subsampled_mat <- as.data.frame(as.matrix(subsampled_mat)) %>% rownames_to_column("gene")
combined_mats <- left_join(mat, subsampled_mat, by = c("gene"))
combined_mats <- as.data.frame(combined_mats) %>%
column_to_rownames("gene") %>%
as.matrix() %>%
as(., "sparseMatrix")
combined_mats[is.na(combined_mats)] <- 0
sobj <- CreateSeuratObject(combined_mats)
new_ids <- sobj@meta.data %>%
rownames_to_column("cell") %>%
mutate(subsampled = ifelse(str_detect(cell, "subsampled"),
"subsampled",
"not subsampled"))
subsampled_cell_ids <- new_ids[new_ids$subsampled == "subsampled", "cell"] %>%
str_replace("::subsampled", "")
new_ids <- mutate(new_ids,
subsampled = ifelse(cell %in% subsampled_cell_ids,
"original cell",
subsampled)) %>%
select(cell, subsampled) %>%
as.data.frame(.) %>%
column_to_rownames("cell")
sobj <- AddMetaData(sobj, new_ids)
sobj <- NormalizeData(sobj)
sobj <- ScaleData(sobj, vars.to.regress = "nUMI")
sobj <- FindVariableGenes(sobj, do.plot = F)
sobj <- RunPCA(sobj, pc.genes = sobj@var.genes, pcs.compute = 20, do.print = F)
sobj <- RunTSNE(sobj, dims.use = 1:18, seed.use = 20180516)
sobj <- FindClusters(sobj, dims.use = 1:18, resolution = 0.6,
print.output = F,
random.seed = 20180516)plt <- TSNEPlot(sobj,
colors.use = brewer.pal(12, "Paired"),
do.label = T) +
labs(title = "PBMCs") +
theme(legend.position = "none")
plt
save_plot("original_with_subsampled_mkcells_tsne.pdf", plt, base_height = 4.25, base_width = 4.25)
subsampled_cells <- TSNEPlot(SetAllIdent(sobj,
"subsampled"),
colors.use = c("lightgrey",
brewer.pal(7, "Set1")[1:2])) +
labs(title = "PBMC") +
theme(legend.position = "bottom")
tmp <- subsampled_cells$data
og_cell_dat <- tmp[cells[[subsampled_libs]], ] %>%
rownames_to_column("cell")
subsampled_cell_dat <- tmp[str_c(cells[[subsampled_libs]], "::subsampled"), ] %>%
rownames_to_column("cell") %>%
mutate(cell = str_replace(cell, "::subsampled", "")) %>%
select(cell, tSNE_1, tSNE_2)
cell_dat <- left_join(og_cell_dat,
subsampled_cell_dat,
by = "cell",
suffix = c("", "_resampled"))
subsampled_cells <- TSNEPlot(SetAllIdent(sobj,
"subsampled"),
colors.use = c("lightgrey",
brewer.pal(7, "Set1")[1:2])) +
geom_segment(data = cell_dat,
aes(x = tSNE_1,
y = tSNE_2,
xend = tSNE_1_resampled,
yend = tSNE_2_resampled),
linejoin = "mitre",
arrow = arrow(length = unit(0.03, "npc"))) +
theme(legend.position = "bottom")
plt <- plot_grid(plt,
subsampled_cells,
nrow = 2)
plt
save_plot("resampled_tsne.pdf", plt,
nrow = 2, ncol = 1,
base_height = 5.5, base_width = 4.25)kNN analysis
Find the k-nearest neighbors in PCA space
## use combined data from above
data.use <- GetCellEmbeddings(object = sobj,
reduction.type = "pca",
dims.use = 1:20)
## find top 10 nearest neighboors using exact search
knn <- RANN::nn2(data.use, k = 5,
searchtype = 'standard',
eps = 0)
subsampled_idxs <- knn$nn.idx[str_detect(rownames(data.use), "::subsampled"), ]
nn_ids <- as_data_frame(t(apply(subsampled_idxs, 1,
function(x)rownames(data.use)[x])))
colnames(nn_ids) <- c("query_cell", paste0("nearest neighbor ", 1:(ncol(nn_ids) - 1)))
nn_ids